Cytochrome c oxidase from yeast and a variety of other eucaryotes is a transmembranous oligomeric enzyme which has many characteristics -e.g., the possession of hydrophilic (peripheral) mitochondrially-synthesized and hydrophobic (integral) cytoplasmically synthesized subunit polypeptides, a functionally asymmetric and transmembranous orientation, and a phospholipid requirement for enzymatic activity -that make it a useful and unique model system for studying membrane assembly. In this study, the structure and assembly of yeast cytochrome c oxidase will be studied both in vivo and in vitro. Subunit-specific antibodies, covalent cross-linkers, and phospholipid vesicles containing purified subunits will be used to study subunit-subunit association and the disposition of subunits relative to the matrix and cytoplasmic surfaces of the inner mitochondrial membrane. Studies on the assembly of the holoenzyme will include: 1) the characterization of a 55K dalton megapeptide precursor to the four hydrophilic cytoplasmically-made subunits; 2) the identification of the membrane bound intermediates in the proteolytic processing of the 55K dalton megapeptide; 3) an analysis of the role of phospholipids in the incorporation of subunits into the inner mitochondrial membrane and in mediating subsequent subunit-subunit interactions; and 4) the identification and characterization of partially-assembled pre-enzymes within the inner mitochondrial membrane.